Three generations of DNA testing

DQ-alphaTEST STRIPAllele = BLUE DOT

RFLPAUTORADAllele = BAND

Automated STRELECTROPHEROGRAMAllele = PEAK

How do they fare?

□ Discriminating power

Sensitivity

Technical artifacts

Speed□ Mixtures

□ Databasing

Discriminating power □ Sensitivity□ Technical artifacts□ Speed□ MixturesDatabasing ٱ

RFLP DQ-alpha STR

Discriminating power

Sensitivity□ Technical artifacts

Speed□ Mixtures

Databasing

RFLP technology

What we would like for them to look like.

What they often look like.

Incomplete digest

Available Kits for STR Analysis

• Kits make it easy for labs to just add DNA samples to a pre-made mix

• 13 CODIS core loci– Profiler Plus and COfiler (PE Applied Biosystems)

– PowerPlex 1.1 and 2.1 (Promega Corporation)

• Increased power of discrimination– CTT (1994): 1 in 410

– SGM Plus™ (1999): 1 in 3 trillion

– PowerPlex ™ 16 (2000): 1 in 2 x 1017

DNA Quantitation using Slot Blot

AMEL

D3

TH01 TPOX

Penta D

Penta EFGAD21 D18

CSF

D16D7

D13D5VWA D8

PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis

Blood Stain

Overview of Steps Involved in DNA Typing

Genotyping by Comparison to Allelic Ladder

REPEATED DNA• Satellite DNA

– Around chromosomal centromere– Long repeats can be 100s to 1000s bp long

• Minisatellite DNA– VNTR– Medium repeats 10 to 100 bp long

• Microsatellites– STR– Short repeats 2 to 6 bp long

SHORT TANDEM REPEATS

• Easy to amplify

• Both heterozygote alleles amplify well

• Number of repeats highly variable

• Good for identification

• Many different sites

STR NOMENCLATURE

• Simple Repeats– Identical length and sequence

• agat agat agat agat agat

• Compound Repeats– Two or more adjacent simple repeats

• agat agat agat ttaa ttaa ttaa

• Complex Repeats– Variable unit length & possible intervening seq

• agat agat aggat agat agat ttaacggccat agat agat

STR NOMENCLATURE

• Microvariants

– Alleles that contain incomplete units

• TH01 9.3

• aatg aatg aatg aatg aatg aatg aatg aatg aatg aatg - 10

• aatg aatg aatg aatg aatg aatg atg aatg aatg aatg - 9.3

STRs Used In Forensic Science

• Need lots of variation - polymorphic

• Overall short segments - 100-400 bp– Can use degraded DNA samples

– Segment size usually limits preferential amplification of smaller alleles

• Single base resolution– TH01 9.3

STRs Used In Forensic Science

• TETRANUCLEOTIDE REPEATS

– Narrow allele size range - multiplexing

– Reduces allelic dropout (stochastic effects)

– Use with degraded DNA possible

– Reduced stutter rates - easier to interpret

mixtures

STR NOMENCLATURE

• Use the 5’ to 3’ (Top) strand

• Start with the first 5’ nucleotide in the repeat

• Microvariants designated by decimal places– Number of complete repeats– Number of bases in incomplete repeat (9.3)

• Allelic ladders used as reference– Contain all common alleles

ALLELIC LADDERS

• Artificial mixture of common alleles• Reference standards• Enable forensic scientists to compare results

– Different instruments– Different detection methods

• Allele quantities balanced • Produced with same primers as test samples• Commercially available in kits

Allelic Ladder Formation

Separate PCR products from varioussamples amplified with primers targetedto a particular STR locus

Combine

Re-amplify

Find representative allelesspanning population variation

Polyacrylamide Gel

Profiler Plus Allelic Ladders

D3S1358 FGAVWA

AMEL D8S1179 D21S11 D18S51

D5S818 D13S317D7S820

ALLELIC LADDERS

THE 13 CODIS STR LOCI

• November 1997

• Average random match probability is greater than 1in 1 trillion

• US slower than UK in selection of loci for database

THE 13 CODIS STR LOCI• Four categories

• Simple repeats– TPOX, CSF1PO, D5S818, D13S317, D16S539

• Simple repeats w/ non-consensus alleles– TH01, D18S51, D7S820

• Compound repeats w/ non-consensus alleles– vWA, FGA, D3S1358, D8S1179

• Complex repeats– D21S11

Commercial STR Kits• Applied Biosystems• AmpFlSTR Profiler Plus

– 5-FAM (BLUE)

– Joe (GREEN)

– NED (YELLOW)

• AmpFlSTR Cofiler

– 5-FAM (BLUE)

– Joe (GREEN)

– NED (YELLOW)

D3S1358 vWA FGA

Amelo D8S1179 D21S11 D18S51

D5S818 D13S317 D7S820

D3S1358 D16S539

Amelo TH01 TPOX CSF1PO

D7S820

Commercial STR Kits• Promega Corporation• PowerPlex 1.1

– Fluorescein (Blue)

– TMR (Yellow)

• PowerPlex 2.1

– Fluorescein (Blue)

– TMR (Yellow)

D5S818 D13S317 D7S820

vWA TH01 TPOX

D3S1358 D21S11

Amelo vWA D8S1179 TPOX FGA

D16S539

CSF1PO

TH01 D18S51 Penta E

Profiler Plus

COfiler

SGM Plus

Green I

Profiler

Blue

TH01

Amel D16S539

D7S820

CSF1POTPOX

D3S1358

D16S539 D18S51D21S11

Amel

Amel

D3S1358

D3S1358

D18S51D21S11

D8S1179

D7S820

D13S317

D5S818

D19S433 D2S1338

FGA

vWA

vWA

FGA

TH01

D3S1358 vWA FGA

D7S820D5S818D13S317

TH01CSF1POTPOX

D8S1179

vWATH01 CSF1PO

TPOXAmel FGAD3S1358

Amel

PCR Product Size (bp) Same DNA Sample Run with Each of the ABI STR Kits

Power of Discrimination1:5000

1:410

1:3.6 x 109

1:9.6 x 1010

1:8.4 x 105

1:3.3 x 1012

D8S1179 D21S11 D7S820 CSF1PO

D3S1358TH01

D13S317 D16S539 D2S1338

D19S433 D18S51TPOX

VWA

AMEL D5S818 FGA

GS500 LIZ size standard

6FAM (blue)

VIC (green)

NED (yellow)

PET (red)

LIZ (orange)

AmpFlSTR® Identifiler™

Genotyping by Comparison to Allelic Ladder

PCR Amplification with Fluorescent STR Kits and Separation with Capillary

Electrophoresis

AMEL

D3

TH01TPOX

Penta D

Penta E

FGAD21 D18

CSF

D16D7

D13D5

VWA D8

Electropherogram

STR LOCI ALLELES• CSF1PO

– c-fms proto-oncogene– Chromosome 5– AGAT repeat– 6 to 15 repeats

• FGA– alpha fibrinogen locus– Chromosome 4– CTTT repeat– 15 to 51.2 repeats

STR LOCI ALLELES• TH01

– TYROSINE HYDROXYLASE– Chromosome 11– TCTA repeat (Bottom strand)– 4 to 11 repeats– Common microvariant 9.3

• TPOX– THYROID PEROXIDASE– Chromosome 2– AATG repeat– 6 to 13 repeats

STR LOCI ALLELES• vWA

– von Willebrand Factor– Chromosome 12– TCTA with TCTG repeat– 10 to 22 repeats

• D3S1358– Chromosome 3– AGAT with AGAC repeat– 12 to 20 repeats

STR LOCI ALLELES• D5S818

– Chromosome 5– AGAT repeat– 7 to 16 repeats

• D7S820– Chromosome 7– GATA repeat– Some Microvariants– 6 to 15 repeats

STR LOCI ALLELES• D8S1179

– Chromosome 8– TCTA repeat with TCTG in alleles >13– 7 to 19 repeats

• D13S317– Chromosome 13– GATA repeat– 7 to 15 repeats

STR LOCI ALLELES• D16S539

– Chromosome 16– AGAT repeat with TCTG in alleles >13– 8 to 15 repeats

• D18S15– Chromosome 18– AGAA repeat– Some x.2 Microvariants– 8 to 27 repeats

STR LOCI ALLELES• D21S11

– Chromosome 21– TCTA repeat with TCTG

– Some x.2 Microvariants – 24 to 38 repeats– Over 70 reported alleles– Fine differences must be sequenced– 4 alleles are designated as 30 repeats

13 CODIS Core STR Loci with Chromosomal Positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

13 CODIS Core STR Loci AMEL

AMEL

Sex-typing

Position of Forensic STR Markers on

Human Chromosomes

Penta E

Penta D

D2S1338

D19S433

An Example Forensic STR Multiplex Kit

D3 FGAvWA 5-FAM (blue)

D13D5 D7 NED (yellow)

A D8 D21 D18 JOE (green)

GS500-internal lane standard

ROX (red)

AmpFlSTR® Profiler Plus™Kit available from PE Biosystems (Foster City, CA)

9 STRs amplified along with sex-typing marker amelogenin in a single PCR

100 bp 400 bp300 bp200 bpSize Separation

Col

or S

epar

atio

n

STR Allele Frequencies

0

5

10

15

20

25

30

35

40

45

6 7 8 9 9.3 10

Caucasians (N=427)

Blacks (N=414)

Hispanics (N=414)

TH01 Marker

*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

Number of repeats

Fre

qu

ency

amelogenin

D19

D3

D8

TH01

VWA D21FGA

D16D18 D2

amelogeninD19

D3D8 TH01

VWA D21

FGA

D16D18 D2

Tw

o di

ffer

ent i

ndiv

idua

ls

DNA Size (base pairs)

Results obtained in less than 5 hours with a spot of blood the size of a pinhead

probability of a random match: ~1 in 3 trillion

Human Identity Testing with Multiplex STRs

Simultaneous Analysis of 10 STRs and Gender

AmpFlSTR® SGM Plus™ kit

Exclusions don’t require numbers Matches do require statistics

Hardy - Weinberg Equilibrium

A1 A2

A1

A2 A1A2

A1A2

A2A2

A1A1

A1A1 A1A2 A2A2

freq(A1) = p1

freq(A2) = p2

p12 p2

22p1p2

p12 p1p2

p1p2 p22

(p1 + p2 )2 = p12 + 2p1p2 + p2

2

A Hardy-Weinberg Population

• LARGE POPULATION

• NO NATURAL SELECTION

• NO MUTATION

• NO IMMIGRATION / EMIGRATION

• RANDOM MATING

Estimate genotype frequency:

1. Frequency at each locus

2. Frequency across all loci

Product Rule

Product Rule • The frequency of a multi-locus STR

profile is the product of the genotype frequencies at the individual loci

ƒ locus1 x ƒ locus2 x ƒ locusn = ƒcombined

Criteria for Use of Product Rule

Inheritance of alleles at one locus have no effect on alleles inherited at other loci

Population database• Look up how often each allele occurs at the

locus in a population (the “allele” frequency)

Item D3S1358 D16S539 TH01 TPOX CSF1P0 D7S820

Q1 16,16 10,12 8,9.3 9,10 12,12 8,11

Item D3S1358 vWA FGA D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820

Q1 16,16 15,17 21,22 13,13 29,30 16,20 8,12 12,12 8,11

CoFIler

ProfIler Plus

D3S1358 = 16, 16 (homozygote)

Frequency of 16 allele = ??

D3S1358 = 16, 16 (homozygote)

Frequency of 16 allele = 0.3071

When same allele:

Frequency = genotype frequency (p2)(for now!)

Genotype freq = 0.3071 x 0.3071 = 0.0943

Item D3S1358 D16S539 TH01 TPOX CSF1P0 D7S820

Q1 16,16 10,12 8,9.3 9,10 12,12 8,11

Item D3S1358 vWA FGA D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820

Q1 16,16 15,17 21,22 13,13 29,30 16,20 8,12 12,12 8,11

CoFIler

ProfIler Plus

VWA = 15, 17 (heterozygote)

Frequency of 15 allele = ??

Frequency of 17 allele = ??

VWA = 15, 17 (heterozygote)

Frequency of 15 allele = ??Frequency of 17 allele = ??

VWA = 15, 17 (heterozygote)

Frequency of 15 allele = 0.2361

Frequency of 17 allele = 0.1833

When heterozygous: Frequency = 2 X allele 1 freq X allele 2 freq

(2pq)

Genotype freq = 2 x 0.2361 x 0.18331 = 0.0866

Overall profile frequency = Frequency D3S1358 X Frequency vWA 0.0943 x 0.0866 = 0.00817

What if…

We encounter alleles not represented in the population database…

…or alleles that are extremely rare in the database???

The Power of Exclusion of a genetic locus is based on the 1 – the sum of squares of all the expected phenotypes/genotypes!

Where the random match probability is the sum of the squares of the observed

phenotype/genotype frequencies in a database,

These measures tell us two things about our markers and databases:

Power of Discrimination – how powerful our loci are

at individualizing

Power of Exclusion – how powerful our marker panel is

at excluding particular genotypes

FBI’s CODIS DNA Database

Combined DNA Index System

• Launched October 1998• Used for linking serial crimes and

unsolved cases with repeat offenders• Links all 50 states• Requires >4 RFLP markers and/or 13

core STR markers• Current backlog of > 600,000 samples

As of June, 2004• Total profiles = 1,857,093• Total forensic profiles = 85,477• Total convicted offender = 1,771,616